Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 June - 20 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lakes and inland sea, and return sludge from sewage plants). Activated sludge was prepared and was controlled in the CERI laboratory. The activated sludge which was cultivated for 21.3 hours after feeding with the synthetic sewage (glucose, peptone and potassium dihydrogenphosphate dissolved in purified in water at an adjusted pH 7.0±1.0) was used.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Reference substance:

aniline

Parameter:

% degradation (test mat. analysis)

Value:

12

Sampling time:

28 d

Remarks on result:

other: based on gc analysis of parent test item (sum of 3 peaks)

Key result

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

28 d

After the end of the incubation, pH of the test solutions was not measured because the test item seemed to be volatile. At the start and end of experiment the test item was found to be insoluble and test solution appears to be colourless.

 

Table 5.2.1/1: Biodegradability of test item after 28 days

	Water + Test item		Sludge + Test item		Average
	Vessel No. 1	Vessel No. 2	Vessel No. 3	Vessel No. 4
BOD* (mg)	0.9	0.9	3.6	4.8	-
Residual amount of test item (mg)	28.1	27.3	24.9	22.0	-
% Biodegradation by BOD	-	1	5	6	4
% Biodegradation by GC	-	3	11	22	12

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In a GLP study biodegradability of test item was evaluated according to OECD 301C "Ready Biodegradability: Modified MITI Test (l)". The average percentage ultimate biodegradation (measured as BOD) was 4% after 28 days. Thus, test item is regarded as not readily biodegradable according to this test.

Executive summary:

In a GLP study biodegradability of test item was evaluated according to OECD 301C "Ready Biodegradability: Modified MITI Test (l)".

 

Test item was tested at the concentration of 100 mg/L along with the activated sludge (30 mg/L)at 25 ± 1°C for the duration of 28 days. Biochemical oxygen demand (BOD) was evaluated using a closed system oxygen consumption measuring apparatus and gas chromatography (GC). Percentage biodegradation by BOD (average) of test item was 4% and Percentage biodegradation of test item by GC (average) was 12 %. Validity criteria for the test were met.

 

Thus, test item is regarded as not readily biodegradable according to this test.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 October 2013 - 10 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Not applicable

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

GLP statement

Inoculum or test system:

natural water: freshwater

Details on inoculum:

Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used.

The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.

Duration of test (contact time):

69 d

Initial conc.:

30 mg/L

Parameter followed for biodegradation estimation:

O2 consumption

Reference substance:

benzoic acid, sodium salt

Remarks:

purity: min. 99.0 %

Key result

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

69 d

Remarks on result:

other: replicate D1

Key result

Parameter:

% degradation (O2 consumption)

Value:

35

Sampling time:

69 d

Remarks on result:

other: replicate D2

Key result

Parameter:

% degradation (O2 consumption)

Value:

20

Sampling time:

69 d

Remarks on result:

other: mean of D1 and D2

Oxygen uptakes, as read on the Oxitop meters, are corrected to account for the small differences between actual and nominal concentrations of test and reference substances.

Degradation of sodium benzoate exceeded 40 % after 7 days and 65 % after 14 days: the activity of the inoculum was thus verified (validity criterion).

Table 5.2.1/2: Biodegradability of test substance

Theoretical Oxygen Demand (ThOD): 2.55 mg O₂/ mg

Days		7	14	21	28	35	42	69
O2uptake of sludge (inoculum blank)	23 B1100	54.0	71.4	81.1	86.8	94.6	98.4	106.0
	24 B2100	52.0	69.3	78.9	86.6	92.4	96.2	108.0
	Mean B100	53.0	70.4	80.0	86.7	93.5	97.3	107.0
O2uptake of substance + sludge	21 C1	48.4	63.9	75.5	81.3	91.0	96.8	110.0
	22 C2	50.6	68.1	75.8	85.6	103.0	111.0	134.1
O2uptake of substance	C1-B100	-4.6	-6.5	-4.5	-5.4	-2.5	-0.5	3.0
	C2-B100	-2.4	-2.3	-4.2	-1.1	9.5	13.7	27.1
% biodegradation of test substance	D1	-6	-8	-6	-7	-3	-1	4
	D2	-3	-3	-6	-1	12	18	35
	Mean D	-5	-6	-6	-4	5	9	20

Calculations

B1₁₀₀, B2₁₀₀-C1 C2 experimental O₂uptake values

B1₁₀₀= (B1₁₀₀+ B2₁₀₀)

D1 = 100 (C1 - B1₁₀₀)/ ThOD * [S]

D1 = 100 (C2 - B1₁₀₀)/ ThOD * [S]

D = (D1 + D2)/2

[S] = Initial test substance concentration (mg/L)

 

Table 5.2.1/2: Biodegradability of Reference substance

Theoretical Oxygen Demand (ThOD): 1.67 mg O₂/ mg

Days		5	7	14	21	28	69
O2uptake of sludge (inoculum blank)	3 B130	16.8	18.9	25.2	27.4	27.4	33.7
	4 B230	17.1	19.3	25.7	27.9	27.9	34.3
	Mean B30	17.0	19.1	25.5	27.7	27.7	34.0
O2uptake of substance + sludge	1 A1	129.0	141.0	162.0	169.0	173.0	190.0
	2 A2	126.0	134.0	156.0	166.0	173.0	185.0
O2uptake of substance	A1-B30	112.1	121.9	136.6	141.4	145.4	156.0
	A2-B30	109.1	114.9	130.6	138.4	145.4	151.0
% biodegradation of test substance	D1	67	73	82	85	87	94
	D2	65	69	78	83	87	91
	Mean D	66	71	80	84	87	92

Calculations

B1₃₀, B2₃₀-A1 A2 experimental O₂uptake values

B1₃₀= (B1₃₀+ B2₃₀)

D1 = 100 (C1 - B1₃₀)/ ThOD * [S]

D1 = 100 (C2 - B1₃₀)/ ThOD * [S]

D = (D1 + D2)/2

[S] = Initial test substance concentration (mg/L)

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

The inherent Biodegradability of test item was determined by the Manometric Respirometry Test according to the OECD Guidelines for Testing of Chemicals, Method No. 302C. At the test concentration test item did not significantly inhibit the intrinsic respiration of the inoculum. Thus, test item should be regarded as not inherently biodegradable according to this test.

Executive summary:

The inherent Biodegradability of test item was determined by the Manometric Respirometry Test according to the OECD Guidelines for Testing of Chemicals, Method No. 302C.

 

Based on biological oxygen demand, in the test conditions test item undergoes no biodegradation after 28 days (4% and 35% after 69 days on the two replicate flasks, respectively).

 

At the test concentration test item did not significantly inhibit the intrinsic respiration of the inoculum.

 

Thus, test item should be regarded as not inherently biodegradable according to this test.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June - 03 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

GLP statement

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Reference substance:

benzoic acid, sodium salt

Remarks:

99% purity

Key result

Parameter:

% degradation (O2 consumption)

Value:

7

Sampling time:

28 d

Results with reference substance:

Degradation of sodium benzoate exceeded 40% after 7 days and 65% after 14 days: the activity of the inoculum was thus verified (validity criterion).

Oxygen uptakes, as read on the Oxitop meters, are corrected to account for the small differences between actual and nominal concentrations of test and reference substances.

Degradation of sodium benzoate exceeded 40 % after 7 days and 65 % after 14 days: the activity of the inoculum was thus verified (validity criterion). The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test substance. Therefore, the test is considered valid.

 

Table 5.2.1/2: Biodegradability of test substance

Theoretical Oxygen Demand (ThOD): 2.55 mg O₂/ mg

Days		5	7	14	21	28	58
O2uptake of sludge (inoculum blank)	3 B1	21.0	23.1	29.5	29.5	31.6	33.7
	4 B2	15.0	17.1	23.6	23.6	25.7	30.0
	Mean B	18.0	20.1	26.6	26.6	28.7	31.9
O2uptake of substance + sludge	11 C1	21.4	23.6	27.9	32.1	34.3	34.3
	12 C2	24.8	24.8	28.9	33.0	33.0	37.2
O2uptake of substance	C1-B	3.4	3.5	1.4	5.6	5.7	2.5
	C2-B	6.8	4.7	2.3	6.4	4.3	5.3
% biodegradation of test substance	D1	4	5	2	7	7	3
	D2	9	6	3	8	6	7
	Mean D	7	5	2	8	7	5

Calculations

B1, B2, C1, C2, A1, A2, E1 experimental O₂uptake values

B1= (B1 + B2)

D1 = 100 (C1 - B)/ ThOD * [S]

D1 = 100 (C2 - B)/ ThOD * [S]

D = (D1 + D2)/2

[S] = Initial test substance concentration (mg/L)

 

Table 5.2.1/2: Biodegradability of Reference substance

Theoretical Oxygen Demand (ThOD): 1.67 mg O₂/ mg

Days		5	7	14	21	28	55
O2uptake of sludge (inoculum blank)	3 B1	21.0	23.1	29.5	29.5	31.6	33.7
	4 B2	15.0	17.1	23.6	23.6	25.7	30.0
	Mean B	18.0	20.1	26.6	26.6	28.7	31.9
O2uptake of substance + sludge	1 A1	131.0	141.0	162.0	171.0	177.0	188.0
	2 A2	132.0	143.0	162.0	170.0	175.0	185.0
O2uptake of substance	A1-B	113.0	120.9	135.5	144.5	148.4	156.2
	A2-B	114.0	122.9	135.5	143.5	146.4	153.2
% biodegradation of test substance	D1	68	73	81	87	89	94
	D2	68	74	81	86	88	92
	Mean D	68	73	81	86	88	93

Calculations

B1, B2, C1, C2, A1, A2, E1 experimental O₂uptake values

B1= (B1 + B2)

D1 = 100 (A1 - B)/ ThOD * [S]

D1 = 100 (A2 - B)/ ThOD * [S]

D = (D1 + D2)/2

[S] = Initial test substance concentration (mg/L)

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Ethyl Safranate undergoes 7% biodegradation after 28 days in the test conditions.

Ethyl Safranate did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

Thus, Ethyl Safranate should be regarded as not readily biodegradable according to this test.

Executive summary:

The Ready Biodegradability of Ethyl Safranate was determined by the Manometric Respirometry Test. Ethyl Safranate undergoes 7% biodegradation after 28 days in the test conditions.

Ethyl Safranate did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

Thus, Ethyl Safranate should be regarded as not readily biodegradable according to this test.

Description of key information

The ready biodegradability of Ethyl Safranate has been investigated using two standard ready screening methods, the OECD 301F Manometric Respirometry Test (Givaudan 13 -E097, key study) and the OECD 301C modified MITI test (CERI 2016, supporting study). The test guideline 301C specifies use of activated sludge precultured with synthetic sewage containing glucose and peptone. This pre-treatment may seriously lower the diversity and biodegradation capacity of the microbes. In contrast the 301F study uses activated sludge directly from wastewater treatment plants. For this reason the 301F study is considered to be the key study and the 301C study as supporting. The average level of biodegradation (measured as BOD) was respectively 7% and 4% after 28 days. Thus, Ethyl Safranate is regarded as not readily biodegradable according to both tests.

 

Specific chemical analysis is obligatory in the MITI method and can be used to assess primary degradation of the test substance. The average percentage primary degradation of the test item (by GC, total peak area) after 28 days was 12% (based on 3 replicates; 3, 11 and 22%). A day 28 extract was subsequently analysed under the conditions of the OECD117 HPLC Partition Coefficient method. The log Pow determined for the converted products varied between 1.3 and 2.5, demonstrating them to be significantly more polar than the parent (log Pow 3.4). Based on LC-MS analysis, some of the converted products were tentatively identified as di- and tri-hydroxylated derivatives of the test item. 

 

In an inherent study 302C study (Givaudan 13 -E169), Ethyl Safranate achieved no biodegradation after 28 days but after 69 days the level of biodegradation (measured as BOD) in the two replicate flasks was 4 and 35% respectively. When assessing inherent biodegradability it is not necessary to define limits on test duration. Biodegradation above 20% may be regarded as evidence of inherent, primary biodegradation. Thus, the results of one replicate indicate that Ethyl Safranate is inherently primarily biodegradable. The different behaviour between the two replicate flasks is probably due to some heterogeneity in the inoculum leading to different microorganism population growth and diversity (i.e. one or more competent microorganism(s) required for the biodegradation to continue were either not introduced into the first flask or had not established themselves to a sufficient concentration for metabolism to be observed). In environmental waters there will be greater homogeneity and biodiversity of the microbes present. Thus under more environmentally realistic test conditions, all replicates would be expected to show some degradation.

Based on the above information, the registered substance is regarded as not readily biodegradable for the purposes of classification and labelling and the conclusion for the exposure assessment is that no biodegradation was observed. However, the indication of primary degradation is taken into consideration for the purposes of the PBT assessment.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater